The endoplasmic reticulum, the first organelle in the secretory pathway, carries out a multiplicity of functions including folding and glycosylation of newly-synthesized polypeptides, assembly of multimeric proteins, storage of Ca++ ions and packaging of secretory proteins into vesicles that are targeted to the next organelle in the secretory chain. The long-term goal of the investigators work is to understand how each of these individual functions is accomplished and how they are coordinated in a way that allows them to be carried coherently. The proposed work couples the advantages of two experimental eukaryotic systems: mammalian cells (or in vitro systems derived from them) for biochemical analysis, and the yeast Saccharomyces cerevisae for genetic analysis. The investigators specific aims are: to analyze the relationship between the structure and function of BiP, the chief chaperone protein of the ER. Using the recently-elucidated three-dimensional structure of N-terminal domain of a closely-related protein (hsc70) as a guide, the investigators will analyze the biochemical and physiological properties of a series of site-directed mutants that (i) alter the amino acids involved in ATP binding and hydrolysis (ii) prevent movement of the hinge region of the molecule (iii) eliminate Ca++ binding sites (iv) modify the sequences within the substrate recognition domain. To analyze in detail the physiological and biochemical properties of a novel form of peptidyl prolyl isomerase (sig-PPI) that appears to be located in the ER of yeast. To explore the role of Ca++ in the maintenance of ER integrity and function. In particular, Dr. Sambrook will investigate the biochemical and physiological behavior of yeast cells carrying mutations in genes coding for three proteins (Ca++ ATPase, calreticulin, and the receptor for Ins (1,4,5)P3 that are thought to be involved in the storage of Ca++ in the ER and in the flux of Ca++ across the ER membrane.
| Mori, K; Sant, A; Kohno, K et al. (1992) A 22 bp cis-acting element is necessary and sufficient for the induction of the yeast KAR2 (BiP) gene by unfolded proteins. EMBO J 11:2583-93 |
| Shubeita, H E; Sambrook, J F; McCormick, A M (1987) Molecular cloning and analysis of functional cDNA and genomic clones encoding bovine cellular retinoic acid-binding protein. Proc Natl Acad Sci U S A 84:5645-9 |
